Free-solution response function interferometry

ABSTRACT

Disclosed are methods for the free solution measurement of molecular interactions by refractive index sensing other than backscattering interferometry. The disclosed methods can have very low detection limits and/or very low sample volume requirements. Also disclosed are various biosensor applications of the disclosed techniques. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit of U.S. Provisional Application No.62/288,926, filed on Jan. 29, 2016, which is incorporated herein byreference in its entirety.

ACKNOWLEDGEMENT

This invention was made with government support under Grant No. CHE1307899 awarded by the National Science Foundation. The government hascertain rights in the invention.

BACKGROUND

Contemporary assays have enabled single molecule detection (Betzig andChichester (1993) Single Molecules Observed by near-Field ScanningOptical Microscopy. Science 262(5138):1422-1425; Levene et al. (2003)Zero-mode waveguides for single-molecule analysis at highconcentrations. Science 299(5607):682-686) have accelerated thesequencing of the human genome (Anonymous (2001) Unsung Heroes. Science291(5507):1207) and facilitated imaging with extraordinary resolutionwithout labels (Hell SW & Wichmann J (1994) Breaking the DiffractionResolution Limit by Stimulated-Emission—Stimulated-Emission-DepletionFluorescence Microscopy. Opt Lett 19(11):780-782). To most closely studyan interaction in the natural state, an assay would interrogate theprocesses (reaction, molecular interaction, protein folding event, etc.)without perturbation. Label-free chemical and biochemical investigations(Liedberg et al. (1995) Biosensing with Surface-Plasmon Resonance—How ItAll Started. Biosens Bioelectron 10(8):R1-R9; Yu et al. (2014) Sheddingnew light on lipid functions with CARS and SRS microscopy. Bba-Mol CellBiol L 1841(8):1120-1129) transduce the desired signal without anexogenous label (fluorescent, radioactive, or otherwise) representing anessential step toward this goal. Many label-free methods require one ofthe interacting species to be either tethered or immobilized to thesensor surface, introducing a potential perturbation to the naturalstate of the species (Moreira et al. (2005).

However, back-scattering interferometry (BSI) is a free-solutionlabel-free technique with the added benefit of sensitivity that rivalsfluorescence (Bornhop et al. (2007) Free-solution, label-free molecularinteractions studied by back-scattering interferometry. Science317(5845):1732-1736). There are other techniques performed in freesolution, such as mass spectrometry (MS) (Cubrilovic et al. (2014)Quantifying Protein-Ligand Binding Constants Using ElectrosprayIonization Mass Spectrometry: A Systematic Binding Affinity Study of aSeries of Hydrophobically Modified Trypsin Inhibitors. J Am Soc MassSpectr 23(10):1768-1777; Kaltashov et al. (2012) Advances and challengesin analytical characterization of biotechnology products: Massspectrometry-based approaches to study properties and behavior ofprotein therapeutics. Biotechnol Adv 30(1):210-222) and nuclear magneticresonance (NMR) (Hu et al. (2004) The mode of action of centrin—Bindingof Ca2+ and a peptide fragment of Karlp to the C-terminal domain. J BiolChem 279(49):50895-50903; Tzeng and Kalodimos (2011) Protein dynamicsand allostery: an NMR view. Curr Opin Struc Biol 21(1):62-67) and thewidely used isothermal titration calorimetry (ITC) (Ababou and Ladbury(2007) Survey of the year 2005: literature on applications of isothermaltitration calorimetry. Journal of Molecular Recognition 20(1):4-14;Liang, Y. (2006) Applications of isothermal titration calorimetry inprotein folding and molecular recognition. J Iran Chem Soc3(3):209-219). As with NMR, ITC has many advantages, but exhibits modestsensitivity and often requires large sample quantities. Anotherincreasingly popular free-solution approach is micro-scalethermophoresis (MST). Yet, for MST to operate label-free, one of thebinding partners must have a significant absorption/fluorescencecross-section (Wienken et al. (2010) Protein-binding assays inbiological liquids using microscale thermophoresis. Nat Commun 1; Zhanget al. (2014) Microscale thermophoresis for the assessment of nuclearprotein-binding affinities. Methods Mol Biol 1094:269-276). BSIrepresents an attractive alternative to these methods because of itshigh sensitivity, small sample volume requirement, optical simplicityand broad applicability (Baksh et al. (2011) Label-free quantificationof membrane-ligand interactions using backscattering interferometry. NatBiotechnol 29(4):357-360; Kussrow et al. (2012) Interferometric Methodsfor Label-Free Molecular Interaction Studies. Anal Chem 84(2):779-792;Olmsted et al. (2014) Toward Rapid, High-Sensitivity, Volume-ConstrainedBiomarker Quantification and Validation using BackscatteringInterferometry. Anal Chem 86(15):7566-7574; Saetear et al. (2015)Quantification of Plasmodium-host protein interactions on intact,unmodified erythrocytes by back-scattering interferometry. Malaria J14). Whereas ITC and MST have well known or established theoreticaldescriptions, the fundamental mechanistic basis for the signal observedin BSI is less well understood.

Accordingly, there remains a need in the art for systems and methods forfree-solution, label-free detection of intermolecular interactionsbetween analytes, preferably with low detection limits and/or low samplevolume requirements.

SUMMARY

As embodied and broadly described herein, the invention, in one aspect,relates to free-solution analytical methods for use in the detection ofmolecular interactions between non-immobilized analytes and/orcharacteristic properties of a sample.

Disclosed are free-solution analytical methods comprising detectingmolecular interactions between a first non-immobilized analyte and asecond non-immobilized analyte, wherein the detection is performed byrefractive index sensing other than backscattering interferometry or bycircular dichroism.

Also disclosed are free-solution analytical methods comprising the stepsof: (a) providing a refractive index sensor for reception of a fluidsample to be analyzed; (b) introducing a first sample comprising a firstnon-immobilized analyte to be analyzed and a second sample comprising asecond non-immobilized analyte to be analyzed onto the sensor, whereinthe first analyte is allowed to interact with the second analyte; (c)interrogating the fluid sample with light; (d) detecting the light afterinteraction with the fluid sample, wherein the detected light is notbackscattered; and (e) detecting a molecular interaction between thefirst and second analyte.

Also disclosed are free-solution analytical methods comprising detectingmolecular interactions between a first non-immobilized analyte and asecond non-immobilized analyte, wherein the determination comprisesdetermining the degree of polymerization, protein folding, proteinaggregation, blood oxygenation, the conformational state of an ionchannel or membrane protein, or the hydration state of an ion chanel ormembrane protein, and wherein the determination is performed byrefractive index sensing.

Also disclosed are free-solution analytical methods comprisingdetermining the degree of polymerization, protein folding, proteinaggregation, blood oxygenation, the conformational state of an ionchannel or membrane protein, or the hydration state of an ion chanel ormembrane protein, and wherein the determination is performed byrefractive index sensing.

Also disclosed are systems comprising a refractive index sensor fordetecting molecular interactions between a first non-immobilized analyteand a second non-immobilized analyte, and a pressure change compensator.

Also disclosed are free-solution analytical methods comprising detectinga molecular change, wherein the detection is performed by refractiveindex sensing other than backscattering interferometry.

Unless otherwise expressly stated, it is in no way intended that anymethod or aspect set forth herein be construed as requiring that itssteps be performed in a specific order. Accordingly, where a disclosedmethod or system does not specifically state that the steps are to belimited to a specific order, it is no way intended that an order beinferred, in any respect. This holds for any possible non-express basisfor interpretation, including matters of logic with respect toarrangement of steps or operational flow, plain meaning derived fromgrammatical organization or punctuation, or the number or type ofaspects described in the specification.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects and together withthe description serve to explain the principles of the invention.

FIG. 1A and FIG. 1B show representative block diagrams of arefractometer (1A) and a forward scattering interferometer (1B).

FIG. 2 shows a representative block diagram of a circular dichroismspectrometer.

FIG. 3 shows a representative diagram of exemplary reference and samplecontents.

FIG. 4A and FIG. 4B show representative data illustrating the A-form toB-form transition of the DNA duplex. Specifically, FIG. 4A shows arepresentative CD spectra of the DNA duplex (inset shows the A-form toB-form transition monitored at 270 nm by ellipticity). FIG. 4B shows arepresentative correlation for BSI signal and ellipticity.

FIG. 5A and FIG. 5B show representative data illustrating the predictedversus BSI experimental values for the CaM binding system (5A) and thecorrelation of χ_(exp) and Xmodel of the unsegregated learning set (5B).

FIG. 6A-F show representative data related to the predicted dry/dc ARIU.Plots show the calculated dry/dc for the reference (∘) and test (+)samples for the Concanavalin A-Mannose system (6A) theCalmodulin-Calcineurin system (6D), the predicted dη/dc_(complex) signalcompared with the experimentally observed signal for ConA-mannose (6B)and c=Calmodulin-calcineurin (6E), and the predicted dη/dc_(complex)signal compared with the experimentally observed signal versus productconcentration for ConA-mannose (5C) and Calmodulin-calcineurin (6F).

FIG. 7A and FIG. 7B show representative comparisons of experimental andmodeled dry/dc signal. Specifically, plots showing the experimental BSIsignal in RIU with the calculated dry/dc signal for recoverin bindingCa²⁺ (7A) and carbonic anhydrase II binding dansylamide (7B).

FIG. 8 shows a representative illustration of the procedure for usingBSI to measure a binding affinity.

FIG. 9A-F show representative BSI block diagrams showing the orientationof the beam relative to the chip (7A-C), a representative image of thefringe pattern (7D), a representative line profile of the region ofinterest for a good fringe pattern (7E), and a representative FFTspectrum for that region of interest (ROI) (7F).

FIG. 10A and FIG. 10B show representative fringe patterns with good (7A)and bad (7B) alignment.

FIG. 11A and FIG. 11B show representative images illustrating theoptical modeling of the beam path for BSI. Specifically, FIG. 11A showsa representative image of ten parallel rays impinged on a chip from theright that are allowed to refract and reflect and exit to the right andinterfering. FIG. 11B shows a representative image of a many beamoptical ray trace of a semicircular channel in a microfluidic chip.

FIG. 12A-D show representative ribbon drawings for Calmodulin unbound(PDB: 1CFD) (12A), bound to Calcium (PDB: 1OSA) (12B), bound to M13(PDB: 1CDL) (12C), and bound to TFP (PDB: 1CTR) (12D).

FIG. 13A and FIG. 13B show representative plots showing the correlationof χ_(exp) and χ_(model) when the learning sets are split into small(13A) and large (13B) χ values.

FIG. 14A and FIG. 14B show representative flow diagrams for predictingthe suitable model (small or large) for a binding pair (14A) and forpredicting the model for the entire learning set (14B).

FIG. 15A and FIG. 15B show representative experimental and modeledFreeSRF binding curves for Recoverin-Ca²⁺ (15A) and carbonic anhydraseII-Dansylamide (15B).

FIG. 16A-D show representative data illustrating Cyfra 21-1 binding CK19as measured via a hand-held refractometer. Specifically, FIG. 16A showsa representative image of a hand-held Reichert RI Detector. FIG. 16Bshows representative data illustrating the response of a hand-held RIdetector for glycerol calibration standards. FIG. 16C showsrepresentative data illustrating label-free, free-solution detection ofCyfra 21-1 in PBS using a hand-held RI detector. FIG. 16D showsrepresentative data illustrating the comparison of signal at 50 ng/mLusing a hand-held RI detector and a BSI detector.

FIG. 17A and FIG. 17B show representative images of recoverin before(17A) and after (17B) Ca²⁺ binding.

FIG. 18A and FIG. 18B show representative images of a RI detector (18A)and the flow path within a RI detector (18B).

FIG. 19 shows representative data illustrating the response of a RIdetector for glycerol calibration standards.

FIG. 20 shows representative data illustrating label-free, free-solutiondetection of mannose in buffer using a RI detector.

FIG. 21A and FIG. 21B show representative data illustrating label-free,free-solution detection of benzene sulfonamide in buffer binding to 50nM CAII (21A) and 10 nM CAII (21B) using a RI detector.

FIG. 22A and FIG. 22B show representative data illustrating label-free,free-solution detection of acetazolamide in buffer using a RI detector.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or can be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION

The present invention can be understood more readily by reference to thefollowing detailed description of the invention and the Examplesincluded therein.

Before the present compounds, compositions, articles, systems, devices,and/or methods are disclosed and described, it is to be understood thatthey are not limited to specific synthetic methods unless otherwisespecified, or to particular reagents unless otherwise specified, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only andis not intended to be limiting. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, example methods andmaterials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedherein can be different from the actual publication dates, which mayneed to be independently confirmed.

A. Definitions

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a substrate,” “apolymer,” or “a sample” includes mixtures of two or more suchsubstrates, polymers, or samples, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. It is also understood that there are a number of valuesdisclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance may or may not occur, andthat the description includes instances where the event or circumstanceoccurs and instances where it does not.

As used herein, the term “polymer” refers to a relatively high molecularweight organic compound, natural or synthetic (e.g., polyethylene,rubber, cellulose), whose structure can be represented by a repeatedsmall unit, the monomer (e.g., ethane, isoprene, β-glucose). Syntheticpolymers are typically formed by addition or condensation polymerizationof monomers.

As used herein, the term “copolymer” refers to a polymer formed from twoor more different repeating units (monomer residues). By way of exampleand without limitation, a copolymer can be an alternating copolymer, arandom copolymer, a block copolymer, or a graft copolymer.

As used herein, the term “bioassay” refers to a procedure fordetermining the concentration, purity, and/or biological activity of asubstance.

As used herein, the term “chemical event” refers to a change in aphysical or chemical property of an analyte in a sample that can bedetected by the disclosed systems and methods. For example, a change inrefractive index (RI), solute concentration and/or temperature can be achemical event. As a further example, a biochemical binding orassociation (e.g., DNA hybridization) between two chemical or biologicalspecies can be a chemical event. That is, a chemical event can be theformation of one or more interaction products of the interaction of afirst analyte with a second analyte. As a further example, adisassociation of a complex or molecule can also be detected as an RIchange. As a further example, a change in temperature, concentration,and association/dissociation can be observed as a function of time. As afurther example, bioassays can be performed and can be used to observe achemical event.

As used herein, the terms “equilibrium constant” and “Kc” and “Keq”refer to the ratio of concentrations when equilibrium is reached in areversible reaction. For example, for a general reaction given by theequation:

aA+bB⇄cC+dD,

the equilibrium constant can be expressed by:

$K_{c} = {\frac{{\lbrack C\rbrack^{c}\lbrack D\rbrack}^{d}}{{\lbrack A\rbrack^{a}\lbrack B\rbrack}^{b}}.}$

An equilibrium constant can be temperature- and pressure-dependent buthas the same value, irrespective of the amounts of A, B, C, and D. Aspecific type of equilibrium constant that measures the propensity of alarger object to separate (dissociate) reversibly into smallercomponents is a “dissociation constant” or “Kd.” A dissociation constantis the inverse of an “affinity constant.”

As used herein, the term “dissociation rate” is a concentrationdependent quantity and involves the “dissociation rate constant” or“K_(D).” The dissociation rate constant relates the rate at whichmolecules dissociate to the concentration of the molecules. Adissociation can be described as AB→A+B, and the rate of dissociation(dissociation rate) is equal to K_(D)[AB]. In general, the larger thevalue of K_(D), the faster the inherent rate of dissociation.

As used herein, the term “association rate” is a concentration dependentquantity and involves the “association rate constant” or “K_(A).” Theassociation rate constant relates the rate at which molecules associateto the concentration of the molecules. An association can be describedas A+B→AB, and the rate of association (association rate) is equal toK_(A)[A][B]. In general, the larger the value of K_(A), the faster theinherent rate of association.

As used herein, the term “free-solution” refers to a lack of surfaceimmobilization. The term is not meant to exclude the possibility thatone or more molecules or atoms of analyte may associate with a surface.Rather, the term can describe the detection of an analyte without therequirement for surface immobilization during analysis.

As used herein, the terms “label-free” and “unlabeled” describe adetection method wherein the detectability of an analyte is notdependent upon the presence or absence of a detectable label. Forexample, “label-free” can refer to the lack of a detectable label. It isunderstood that the ability of a label to be detected can be dependentupon the detection method. That is, an analyte having a moiety capableof serving as a detectable label for a first detection method can beconsidered “label-free” or “unlabeled” when a second detection method(wherein the label is not detectable) is employed. In a further aspect,the analytes employed in the disclosed systems and methods can lackdetectable labels.

As used herein, the term “detectable label” refers to any moiety thatcan be selectively detected in a screening assay. Examples includewithout limitation, radiolabels (e.g., ³H, ¹⁴C, ³⁵S, ¹²⁵I, ¹³¹I)affinity tags (e.g. biotin/avidin or streptavidin), metal bindingdomains, epitope tags, FLASH binding domains (see U.S. Pat. Nos.6,451,569; 6,054,271; 6,008,378 and 5,932,474), glutathione or maltosebinding domains, photometric absorbing moieties, fluorescent orluminescent moieties (e.g. fluorescein and derivatives, GFP, rhodamineand derivatives, lanthanides etc.), and enzymatic moieties (e.g.horseradish peroxidase, β-galactosidase, β-lactamase, luciferase,alkaline phosphatase). Such detectable labels can be formed in situ, forexample, through use of an unlabeled primary antibody which can bedetected by a secondary antibody having an attached detectable label.Further examples include imaging agents such as radioconjugate,cytotoxin, cytokine, Gadolinium-DTPA, a quantum dot, iron oxide, andmanganese oxide.

Disclosed are the components to be used to prepare the compositions ofthe invention as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc., of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds may not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular compound is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the compound are discussed, specifically contemplated is eachand every combination and permutation of the compound and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the compositions of the invention. Thus, if there are avariety of additional steps that can be performed it is understood thateach of these additional steps can be performed with any specific aspector combination of aspects of the methods of the invention.

It is understood that the compositions disclosed herein have certainfunctions. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures that can perform the same function that arerelated to the disclosed structures, and that these structures willtypically achieve the same result.

B. Refractive Sensing

In one aspect, disclosed are systems comprising a refractive indexsensor for detecting molecular interactions between a firstnon-immobilized analyte and a second non-immobilized analyte, and apressure change compensator. In a further aspect, both analytes areunlabeled. In a still further aspect, at least one of the analytes ispresent in an amount of less than about 1.0×10⁻³ M.

In various aspects, refractive sensing refers to the measurement of therefractive index of a sample, for example, a fluid sample. Therefractive index or change in refractive index of a sample can be usedto determine a variety of biologically important measurements including,but not limited to, an equilibrium constant, a dissociation constant, adissociation rate, an association rate, a concentration of an analyte,and the presence of an analyte. Refractive index measurements are alsoused in other applications such as, for example, process control and thedetection of explosives (Bowen et al. (2003) “Gas phase detection oftrinitrotoluene utilizing a solid-phase antibody immobilized on a goldfilm by means of surface plasmon resonance spectroscopy” Appl.Spectrosc. 57(8): 906-914).

Various devices and techniques for measuring refractive index are known.These include the Abbe-type refractometer (see FIG. 1A), and sensorsbased on surface plasmon resonance. Optical waveguides can also be used.The presence of a liquid adjacent to an optical waveguide can alter theeffective modal index of light propagating within the waveguide. Thismodification of index can be measured using techniques that aresensitive to changes in optical path length. For example, interferometerstructures have been used to measure index changes and hence to sensethe presence of proteins (Heideman et al. (1993) “Performance of ahighly sensitive optical wave-guide Mach-Zehnder interferometerimmunosensor” Sensors and Actuators B-Chemical 10(3): 209-217) (see FIG.1B).

Referring to FIG. 1A, a block diagram of an exemplary Abbe-typerefractometer is illustrated. As shown, the sample is contained betweentwo prisms, the illuminating prism and the measuring prism. The lightsource generates light, which enters the sample from the illuminatingprism. The surface of the illuminating prism is matted, so that lightenters the sample at all possible angles, including those almostparallel to the surface. The light is then refracted at the criticalangle at the bottom surface of the measuring prism and directed into thetelescope. Additionally, two Amici prisms that can be rotated arelocated within the telescope (not shown), which can be used to correctthe dispersion. The telescope is used to measure the position of theborder between dark and light areas. Knowing the angle and refractiveindex of the measurement prism allows for the refractive index of thesample to be calculated.

Referring to FIG. 1B, a block diagram of an exemplary Michelsoninterferometer is illustrated. As shown, a coherent light source emitslight that hits a beam splitter. A portion of the light is transmitteddirectly through beam splitter to mirror A, while some is reflected inthe direction of mirror B. Both beams are then reflected back onto thebeam splitter to produce an interference pattern incident on thedetector. If an angle is observed between the two returning beams thedetector will record a sinusoidal fringe pattern. Alternatively, ifthere is perfect spatial alignment between the two returning beams, thedetector will record a constant intensity over the beam dependent on thedifferential path length.

Accordingly, the disclosed invention provides a method for detectingmolecular interactions between a first non-immobilized analyte and asecond non-immobilized analyte, wherein the detection is performed byrefractive index sensing other than backscattering interferometry.Examples of refractive index (RI) detectors include, but are not limitedto, RI detectors based on the angle of deviation method of measurement,RI detectors based on the Fresnel method of RI measurement, aChristiansen effect detector, an interferometer detector, or adifferential refractometer detector. Additional examples include arefractomax 521 RI detector, a RID-20A RI detector, a RID-10A RIdetector, a Waters RI detector, a Waters RI detector, a Wyatt RIdetector, a HPLC, an Acquity RI detector, a 1260 Infinity RI detector,an Optilab RI detector, a Knauer RI detector, a Shimadzu RI detector, aShodex RI detector, a LC-4000 Series RI detector, or other suitablysensitive RI detectors.

In a further aspect, detection is performed by refractive index sensingother than forward scattering or side scattering interferometry.

In various aspects, the first and/or second analyte may be contained in,e.g. flowed through, a capillary dimensioned flow channel such as acapillary tube. The cross-sectional depth of the channel is limited onlyby the coherence length of the light and its breadth is limited only bythe width of the light beam. Preferably, the depth of the channel isfrom 1 to 10 μm, but it may be from 1 to 20 μm or up to 50 μm or more,e.g. up to 1 mm or more. However, sizes of up to 5 mm or 10 mm or moreare possible. Suitably, the breadth of the channel is from 0.5 to 2times its depth, e.g., equal to its depth. In various aspects, thechannel may comprise a substantially circular, generally semi-circular,or rectangular cross-section.

The sample is typically a liquid, and can be flowing or stationary.However, the sample can also be a solid or a gas in various aspects ofthe present invention. The first and/or further materials will normallybe solid but in principle can be liquid, e.g., can be formed by asheathing flow of guidance liquid(s) in a microfluidic device, with thesample being sheathed flow of liquid between such guidance flows. Thesample may also be contained in a flow channel of appropriate dimensionsin substrate such as a microfluidic chip. The method may therefore beemployed to obtain a read out of the result of a reaction conducted on a“lab on a chip” type of device.

The invention includes apparatus for use in performing a method asdescribed, which apparatus comprises a source of spatially coherentlight, a sample holder for receiving a sample upon which to perform themethod positioned in a light path from the light source, a detector fordetecting light, and data processing means for receiving measurements oflight intensity from the detector and for conducting an analysisthereon, wherein the analysis comprised determining an equilibriumconstant, a dissociation constant, a dissociation, rate an associationrate, calculating a change in hydrodynamic volume, entropy, or enthalpy,the concentration of the first and/or second analyte, identifying thepresence of the first and/or second analyte, or identifying the presenceof a third analyte. In various aspects, the analysis comprisesquantification of the sample.

In a further aspect, the RI sensor comprises a channel formed in asubstrate, the channel has a longitudinal direction and a transversedirection, and a light source for generating a light, wherein the lightis elongated in the longitudinal direction of the channel

In various aspects, the channel of the present invention can be formedfrom a substrate such as a piece of silica or other suitable opticallytransmissive material. In various aspects, the material of compositionof the substrate has a different index of refraction than that of thesample to be analyzed. In a further aspect, as refractive index can varysignificantly with temperature, the substrate can optionally be mountedand/or connected to a temperature control device. In a still furtheraspect, the substrate can be tilted, for example, about 7°, such thatscattered light from channel can be directed to a detector.

In a further aspect, the channel has a generally semi-circularcross-sectional shape. A unique multi-pass optical configuration isinherently created by the channel characteristics, and is based on theinteraction of the unfocused laser beam and the curved surface of thechannel that allows interferometric measurements in small volumes athigh sensitivity. Alternatively, the channel can have a substantiallycircular or generally rectangular cross-sectional shape. In a stillfurther aspect, the substrate and channel together comprise a capillarytube. In yet a further aspect, the substrate and channel togethercomprise a microfluidic device, for example, a silica substrate, or apolymeric substrate [e.g., polydimethylsiloxane (PDMS) or polymethylmethacrylate (PMMA)], and an etched channel formed in the substrate forreception of a sample, the channel having a cross sectional shape. In aneven further aspect, the cross sectional shape of a channel issemi-circular. In a still further aspect, the cross sectional shape of achannel is square, rectangular, or elliptical. In yet a further aspect,the cross sectional shape of a channel can comprise any shape suitablefor use in a BSI technique. In an even further aspect, a substrate cancomprise one or multiple channels of the same or varying dimensions. Invarious aspects, the channel can have a radius of from about 5 to about250 micrometers, for example, about 5, 10, 20, 30, 40, 50, 75, 100, 150,200, or 250 micrometers. In still other aspects, the channel can have aradius of up to about 1 millimeter or larger, such as, for example, 0.5millimeters, 0.75 millimeters, 1 millimeter, 1.25 millimeters, 1.5millimeters, 1.75 millimeters, 2 millimeters, or more.

In various aspects, the source of coherent light is a laser, suitably aHe—Ne laser or a diode laser or VCSEL. The laser light may be coupled tothe site of measurement by known wave-guiding techniques or may beconventionally directed to the measurement site by free spacetransmission.

In various aspects, the detected light is representative of therefractive index of the sample. The measured refractive index can beindicative of a number of properties of the sample including, but notlimited to, the presence or concentration of a solute substance, e.g., areaction product, pressure, temperature, or flow rate (e.g., bydetermining when a thermal perturbation in a liquid flow reaches adetector).

In one aspect, the detector is a CCD array of suitable resolution.

The apparatus can comprise means for controlling the temperature of thesample, e.g., a heater and/or a Peltier cooler and a temperaturemeasuring device.

The invention includes apparatus as described herein, wherein the sampleholder is configured to allow a sample to flow there through and whereinthe sample holder is connected to receive a separated sample from asample separation device in which components of a mixed sample areseparated, e.g., by capillary electrophoresis, capillaryelectrochromatography, or HPLC. Accordingly, viewed from anotherperspective, the invention provides chromatography apparatus having arefractive index measuring unit as described herein as a detector.

More generally, the sample holder of the apparatus described above canbe a flow through passage so that the contents of the channel may becontinuously monitored to observe changes in the content thereof. Thesechanges may include the temporary presence of cells and the out flowfrom the sample holder may be diverted to a selected one of two or moreoutlet channels according to the measurements of refractive indexobserved in the sample holder, e.g., to achieve sorting of cells inresponse to such measurements. The sample holder can contain astationary analytical reagent (e.g., a coating of an antibody,oligonucleotide, or other selective binding agent) and changes in therefractive index caused by the binding of a binding partner to thereagent may be observed. In view of the small sample size which it ispossible to observe, the sample holder can contain a biological cell andmetabolic changes therein may be observed as changes in the refractiveindex of the cell.

Thus, in various aspects, the sample solution and the reference solutionmay be picked up individually into a cell such as, for example, acapillary tube. Each sample is then loaded into a tray for reading. In afurther aspect, the tray can have several cells (e.g., capillaries)integrated into it. In this way, the samples are delivered to theindividual cells for introduction into the sensor for analysis.

In one aspect, the invention relates to a method for detecting molecularinteractions between a first non-immobilized analyte and a secondnon-immobilized analyte, wherein the detection is performed byrefractive index sensing other than backscattering interferometry,wherein the detection comprises determining refractive index variationsin the intensity of reflections of light which has passed through thefirst and second analyte. In a further aspect, detection is performed byrefractive index sensing other than forward scattering or sidescattering interferometry.

Refractive index can be negatively affected by changes in pressure.Moreover, as the concentration of the analyte decreases, even smallerchanges in pressure can have a significantly greater impact. Thus, invarious aspects, the system may comprise a pressure change compensator.A pressure change compensator can balance the pressure inside andoutside of the detection system by compensating for variations in thevolume of the liquid within the system, which may be due to variationsin the ambient pressure and/or temperature. Examples of pressurecompensators include, but are not limited to, a back-pressure restrictorand a capillary restrictor.

A change in environmental temperature can also negatively impact a RIdetector. Thus, in various aspects, the system may comprise atemperature change compensator. A temperature change compensator canbalance the temperature inside and outside of the detection system bycompensating for variations in the temperature of the liquid within thesystem, which may be due to, for example, variations in the ambientpressure and/or temperature. Alternatively, the temperature changecompensator can change the temperature of the incoming mobile phase tomatch that of the solvent in the detector. Examples of temperaturecompensators include, but are not limited to, a thermostat cabinet, athermoelectric temperature controller (e.g., Peltier) and a heatexchanger.

C. Circular Dichroism

Circular dichroism (CD) is the difference in the absorption ofleft-handed circularly polarized light (L-CPL) and right-handedcircularly polarized light (R-CPL) and occurs when a molecule containsone or more chiral chromophores (light-absorbing groups). CD has a widerange of applications including, but not limited to, analyzing thestructure of small molecules, DNA, peptides, nucleic acids,carbohydrates, and proteins, identifying charge-transfer transitions,determining geometric and electronic structure, and analyzing molecularinteractions. CD spectra are measured using a circular dichroismspectrometer (see FIG. 2).

Accordingly, the disclosed invention provides a method for detectingmolecular interactions between a first non-immobilized analyte and asecond non-immobilized analyte, wherein the detection is performed bycircular dichroism.

Referring to FIG. 2, a block diagram of an exemplary CD spectrometer isillustrated. As shown, the light source emits light that hits themonochromator (MC). A narrow band of wavelengths then pass through thelinear polarizer (LP), which splits the unpolarized monochromatic beaminto two linearly polarized beams. Next, one of the two linearlypolarized beams passes through the photoelastic modulator (PEM), whichconsists of a plate made of a transparent, optically isotropic materialbonded to a piezoelectric quartz crystal. When an alternating electricfield is applied, the light emerging from the PEM switches from L-CPL toR-CPL and back with the frequency of the applied electric field. If thesample exhibits CD, the amount of light absorbed varies periodicallywith the polarization of the incident light. This, in turn, causes theintensity of the light that reaches the detector to exhibit sinusoidalintensity variations at the frequency of the field applied. Thus, thedetector output consists of a signal with a small alternating current(AC) component superimposed on a direct current (DC) component. The ACcomponent is filtered out and amplified. The ratio of the AC to the DCcomponent is directly proportional to the circular dichroism of thesample, and this quantity is recorded as a function of wavelength toprovide a CD spectrum.

D. Methods for Free-Solution Determination of Molecular Interactions

In contrast to conventional techniques that observe immobilizedanalytes—which necessarily limit conformational and translationalfreedom for analytes and are, thus, in vitro measurements—free-solutionanalysis techniques mimic in vivo measurements, because analytes enjoyunrestricted freedom in all three dimensions during measurement.

In one aspect, disclosed are free-solution analytical methods comprisingdetecting molecular interactions between a first non-immobilized analyteand a second non-immobilized analyte, wherein the detection is performedby refractive index sensing other than backscattering interferometry orby circular dichroism. In a further aspect, detection is performed byrefractive index sensing other than forward scattering or sidescattering interferometry.

In one aspect, disclosed are free-solution analytical methods comprisingthe steps of: (a) providing a refractive index sensor for reception of afluid sample to be analyzed; (b) introducing a first sample comprising afirst non-immobilized analyte to be analyzed and a second samplecomprising a second non-immobilized analyte to be analyzed onto thesensor, wherein the first analyte is allowed to interact with the secondanalyte; (c) interrogating the fluid sample with light; (d) detectingthe light after interaction with the fluid sample, wherein the detectedlight is not backscattered; and (e) detecting a molecular interactionbetween the first and second analyte.

In one aspect, disclosed are free-solution analytical methods comprisingdetecting molecular interactions between a first non-immobilized analyteand a second non-immobilized analyte, wherein the determinationcomprises determining the degree of polymerization, protein folding,protein aggregation, blood oxygenation, the conformational state of anion channel or membrane protein, or the hydration state of an ion chanelor membrane protein, and wherein the determination is performed byrefractive index sensing. In a further aspect, the method comprises thesteps of: (a) providing a refractive index sensor for reception of afluid sample to be analyzed; (b) introducing a first sample comprising afirst non-immobilized analyte to be analyzed and a second samplecomprising a second non-immobilized analyte to be analyzed onto thesensor, wherein the first analyte is allowed to interact with the secondanalyte to form one or more interaction products; (c) interrogating thefluid sample with light; (d) detecting the deflected and/or refractedlight after interaction with the fluid sample, wherein the light is notbackscattered; and (e) detecting a molecular interaction between thefirst and second analyte.

In one aspect, disclosed are free-solution analytical methods comprisingdetermining the degree of polymerization, protein folding, proteinaggregation, blood oxygenation, the conformational state of an ionchannel or membrane protein, or the hydration state of an ion chanel ormembrane protein, and wherein the determination is performed byrefractive index sensing. In a further asepct, refractive index sensingis not via backscattering interferometry.

In a further aspect, the light is not scattered.

In a further aspect, refractive index sensing is via a refractometer. Ina still further aspect, refractive index sensing is via interferometry.In yet a further aspect, refractive index sensing is via forwardscattering interferometry. In an even further aspect, refractive indexsensing is via backscattering interferometry. In a still further aspect,refractive index sensing is via a hand-held refractive index sensingdevice.

It is contemplated that the method can be used to determine, forexample, one or more of an equilibrium constant, a dissociationconstant, a dissociation rate, a dissociation rate constant, anassociation rate, and/or an association rate constant of theinteraction. In a further aspect, the method can be used to determine,for example, the concentration of the first and/or second analyte. In astill further aspect, the method can be used to determine, for example,the presence of the first and/or second analyte. In yet a furtheraspect, the method can be used to determine, for example, the presenceof a third analyte.

Each of the one or more analytes can be introduced onto the sensor in asample. Two or more analytes can be present in the same or in differentsamples. Each of the one or more analytes can independently be presentin a suitable concentration, for example, a concentration of less thanabout 5.0×10⁻⁴M, of less than about 1.0×10⁻⁴M, of less than about5.0×10⁻⁵M, of less than about 1.0×10⁻⁵M, of less than about 5.0×10⁻⁶M,of less than about 1.0×10⁻⁶M, of less than about 5.0×10⁻⁷M, of less thanabout 1.0×10⁻⁷M, a concentration of less than about 5.0×10⁻⁸M, of lessthan about 1.0×10⁻⁸M, of less than about 5.0×10⁻⁹M, of less than about1.0×10⁻⁹M, of less than about 1.0×10⁻¹° M, of less than about 5.0×10⁻¹⁰M of less than about 5.0×10⁻¹¹M, of less than about 1.0×10⁻¹¹M, of lessthan about 5.0×10⁻¹²M, of less than about 1.0×10⁻¹²M, of less than about5.0×10⁻¹³M, of less than about 1.0×10⁻¹³M, of less than about5.0×10⁻¹⁴M, of less than about 1.0×10⁻¹⁴M, of less than about5.0×10⁻¹⁵M, or of less than about 1.0×10⁻¹⁵M.

In one aspect, the interaction can be a biomolecular interaction. Forexample, two analytes can associate to provide an interaction product(e.g., adduct, complex, or new compound). In a still further aspect, ananalyte can dissociate to provide two or more interaction products. Inyet a further aspect, more than two analytes can be involved in theinteraction.

In a further aspect, the first and/or second analyte is a complex. In astill further aspect, the complex is a chemical or biochemical complex.In yet a further aspect, the complex was formed prior to the introducingstep. In an even further aspect, the complex was formed subsequent tothe introducing step.

The disclosed techniques can determine the interaction between one ormore analytes by monitoring, measuring, and/or detecting the formationand/or steady state relative abundance of one or more analyteinteraction products from the interaction of the one or more analytes.The determination can be performed qualitatively or quantitatively.Interaction rate information can be derived from various measurements ofthe interaction.

In a further aspect, the first sample is combined with the second sampleprior to introduction. That is, the analytes are combined (andpotentially interacting) prior to performing the disclosed methods. Inthis aspect, the step of introducing the first sample and the step ofintroducing the second sample are performed simultaneously.

In a further aspect, the first sample is combined with the second sampleafter introduction. That is, the analytes can be combined at a pointbefore the sensor, or at a point on the sensor, when performing thedisclosed methods. In this aspect, the step of introducing the firstanalyte and the step of introducing the second analyte are performedeither simultaneously or sequentially. In a further aspect, thedetecting step is performed during the interaction of the first analytewith the second analyte.

Thus, in various aspects, the first and second samples are introducedsimultaneously. In a further aspect, the first and second samples areintroduced sequentially. In a still further aspect, the first analyte isallowed to interact with the second analyte prior to introducing thefirst and/or second sample onto the sensor. In yet a further aspect, thefirst analyte is allowed to interact with the second analyte afterintroducing the first and/or second sample onto the sensor. In an evenfurther aspect, the first analyte is allowed to interact with the secondact while introducing the first and/or second sample onto the sensor.

A first sample (e.g., a solution including a first non-immobilizedanalyte to be analyzed) can be introduced onto the refractive indexsensor. The first sample can be provided having a known concentration ofthe first analyte. A baseline response can then be established bydirecting light onto the first sample.

A second sample (e.g., a solution including a second non-immobilizedanalyte to be analyzed) can then be introduced onto the refractive indexsensor. In various aspects, the second sample can be provided as apre-mixed sample of the first non-immobilized analyte and the secondnon-immobilized analyte or provided by adding a sample comprising thesecond non-immobilized analyte to the first sample. In one aspect, thefirst sample is a solution of the first analyte, which is displaced onthe sensor by the introduction of the second sample, which is a solutionof both the first analyte and the second analyte. The second sample canbe provided having a known concentration of the first analyte, which canbe the same as the concentration of the first analyte in the firstsolution. The second sample can also be provided having a knownconcentration of the second analyte. Light can then be directed onto thesensor.

In various aspects, a reference sample can be introduced onto therefractive index sensor. The reference sample can be introduced onto therefractive index sensor prior to or subsequent to introduction of thefirst and/or second sample. In a further aspect, the reference samplecomprises a first non-immobilized analyte to be analyzed. In a stillfurther aspect, the method further comprises the steps of: (a)interrogating the reference sample with light; (b) detecting the lightafter interaction with the reference sample, wherein the detected lightis not backscattered; (c) determining a characteristic of the referencesample; and (e) employing the characteristic of the reference sample tocompensate for background interference effects in the determination ofthe molecular interaction between the first and second analyte.Exemplary reference samples are illustrated in FIG. 3.

In various aspects, the reference sample can be a ligand alone in theabsence of the matrix, more often referred to as a blank. Referencesamples are typically comprised of the ligand in the experimental matrix(cell vesicle, cell lysate, serum, urine, other biofluids, etc.) thatdoes not contain the receptor. Examples include, but are not limited to,cell-based matrices that do not contain the receptor, null lipoparticles(devoid of the target receptor), empty viral particle/bacteriophagescaffolds, and an experimental matrix stripped of receptor.

In various aspects, the reference sample can be the experimental matrixcontaining the receptor with a non-binding analyte molecule that issimilar to the ligand. Examples include, but are not limited to, adenatured ligand, an isotype matched antibody for a different compound,a compound with a similar structural backbone as the ligand that hasadditional or removed functional groups (e.g., phenol versus2,4,6-trinitrophenol, dopamine versus 3-methoxytyramine, tyrosine versus3-nitrotyrosine, serotonin versus tryptophan, histamine versushistidine), a nonsense nucleic acid strand of the same length, a nucleicacid stand with >3 base-pair mismatch, a compound of similar size as theligand known to not bind the receptor, and other molecules known tonon-specifically bind to the matrix (e.g., cholesterol for membranebased experiments).

In various aspects, the reference sample can be the ligand with theexperimental matrix containing the receptor (cell-based matrices,tissues-based matrices, serum, urine, other biofluids, etc.) that hasbeen treated to inhibit binding. Examples include, but are not limitedto, enzyme treatment of the receptor, blocking of the receptor with anantibody, blocking of the receptor with a known binding compound,blocking of the receptor with an inhibitor, denaturation of thereceptor, receptor without the cofactors necessary for binding, (e.g.,calmodulin without Ca²⁺, concanavalin A without Mn²⁺ and Ca²⁺, recoverinwithout Ca²⁺, etc.).

In a further aspect, the first analyte and/or the second analyte is/areunlabeled. While the disclosed methods can be used in connection withunlabeled analytes, it is contemplated that the analytes can beoptionally labeled. Such labeling can be convenient for preceding,subsequent, or simultaneous analysis by other analytical methods. In astill further aspect, at least one of the analytes is unlabeled. In yeta further aspect, both analytes are unlabeled.

In a further aspect, the the first and/or second analyte is one or moreof an antibody, an antigen, a protein, a small molecule, a drug, areceptor, a cell, an oligonucleotide, a carbohydrate, an enzyme, asubstrate, a DNA, an aptamer, a RNA, a nucleic acid, a biomolecule, amolecular imprint, a protein mimetic, an antibody derivative, a lectin,a cell membrane, an ion, a virus particle, a bacteria, and a micro-RNA.

In a further aspect, detecting a molecular interaction comprisesdetermining a change in a physical or chemical property of the firstand/or second sample. In a still further aspect, the change in aphysical or chemical property of the first and/or second samplecorresponds to the formation of one or more interaction products.

In a further aspect, detecting a molecular interaction comprisesdetermining an equilibrium constant, a dissociation constant, adissociation rate, or an association rate. In a still further aspect,detecting comprises determining the concentration of the first and/orsecond analyte. In yet a further aspect, detecting comprises identifyingthe presence of a third analyte. In an even further aspect, calculatinga change in hydrodynamic volume, entropy, or enthalpy.

In a further aspect, detecting a molecular interaction comprisesdetermining a change in a physical or chemical property of the fluidsample. In a still further aspect, the change in physical or chemicalproperty of the fluid sample corresponds to the formation of one or moreinteraction products.

In a further aspect, the molecular interaction is the formation of oneor more covalent bonds, electrostatic bonds, hydrogen bonds, orhydrophobic interactions. In a further aspect, the molecular interactionis a binding event between one or more of antibody-antigen,protein-protein, small molecule-small molecule, small molecule-protein,drug-receptor, antibody-cell, virus-cell, virus-protein, bacteria-cell,bacteria-protein, virus-DNA, virus-RNA, bacteria-DNA, bacteria-RNA,protein-cell, oligonucleotide-cell, carbohydrate-cell, cell-cell,enzyme-substrate, protein-DNA, protein-aptamer, DNA-DNA, RNA-DNA,DNA-RNA, protein-RNA, small molecule-nucleic acid, biomolecule-molecularimprint, biomolecule-protein mimetic, biomolecule-antibody derivatives,lectin-carbohydrate, biomolecule-carbohydrate, small molecule-cellmembrane, ion-protein, and protein-protein.

In a further aspect, the molecular interaction comprises one or more ofa change in conformational structure of the first and/or second analyteand a change in hydration of the first and/or second analyte. In a stillfurther aspect, the molecular interaction comprises a change inconformational structure of the first and/or second analyte. In yet afurther aspect, the molecular interaction comprises a change inhydration of the first and/or second analyte. In an even further aspect,the molecular interaction comprises a change in conformational structureof the first and/or second analyte and a change in hydration of thefirst and/or second analyte.

In a further aspect, the molecular interaction lacks a change in mass.In a still further aspect, the molecular interaction is a chemicalreaction.

In various aspects, the first and/or second sample is a fluid sample. Ina further aspect, a fluid sample can comprise at least one of a liquidor a gas. In particular aspects, a fluid sample comprises a solution ofone or more analytes and one or more liquid solvents. A solution can beprovided in an organic solvent or in water. In certain aspects, thesolution can comprise man-made preparations or naturally occurringsubstances. In certain aspects, the solution can comprise a body fluid(e.g., peripheral blood, urine, cerebrospinal fluid, pulmonary lavage,gastric lavage, bile, vaginal secretions, seminal fluid, aqueous humor,vitreous humor, serum, and saliva) from a human, a mammal, anotheranimal, or a plant.

In a further aspect, the refractive index sensor comprises a prism.

In a further aspect, the refractive index sensor comprises a substratehaving a first channel formed therein for reception of a fluid sample tobe analyzed and wherein the first and second analyte are introduced intothe channel. Generally, the substrate and channel can comprise anymaterial suitable for containing and providing a sample for analysis andcapable of being interrogated by light. In one aspect, the substrate andchannel together comprise a capillary tube. In a further aspect, whereinthe substrate and channel together comprise a microfluidic device.

In a further aspect, the microfluidic device comprises a polymericsubstrate and an etched channel formed in the substrate for reception ofa fluid sample, the channel having a cross sectional shape. In a furtheraspect, the polymeric substrate can be selected from rigid andtransparent plastics. In various further aspects, the polymericsubstrate comprises one or more polymers selected from polycarbonate,polydimethylsiloxane, fluorosilicone, polytetrafluoroethylene,poly(methyl methacrylate), polyhexamethyldisilazane, polypropylene,starch-based polymers, epoxy, and acrylics.

In a further aspect, the microfluidic device comprises a silicasubstrate and an etched channel formed in the substrate for reception ofa fluid sample, the channel having a cross sectional shape, which can besubstantially circular, substantially semi-circular, or substantiallyrectangular, as disclosed herein.

In a further aspect, the first channel is configured for reception oftwo or more fluid samples by having at least two inlets positioned atopposing locations of the channel, and at least one outlet positioned ata point between the at least two inlets, thereby defining a right sideof the channel and a left side of the channel and wherein the firstsample is introduced into the right side of the channel and the secondsample is introduced into the left side of the channel.

It is contemplated the substrate can comprise one or more than onechannel. Thus, in a further aspect, the substrate further comprises asecond channel. In a still further aspect, the first analyte isintroduced into the first channel and the second analyte is introducedinto the second channel. In yet a further aspect, the first and secondanalyte are introduced into the first channel.

In a further aspect, the substrate further comprises a referencechannel. In a still further aspect, the method further comprises thesteps of: (a) introducing a reference sample; (b) interrogating thereference sample with light; (c) detecting the light after interactionwith the reference sample, wherein the detected light is notbackscattered; (d) determining a characteristic of the reference sample;and (e) employing the characteristic of the reference sample tocompensate for background interference effects in the determination ofthe molecular interaction between the first and second analyte in thefirst channel.

The disclosed methods can provide real-time, free-solution detection ofmolecular interactions with very low detection limits. That is, in oneaspect, the invention relates to a free-solution analytical method fordetecting molecular interactions comprising the step of detecting amolecular interaction between two non-immobilized analytes, wherein atleast one of the analytes is present during the determination at aconcentration of less than about 5.0×10⁻⁴M. In various furtherembodiments, the concentration can be less than about 1.0×10⁻⁴ M, forexample, less than about 5.0×10⁻⁵M, less than about 1.0×10⁻⁵M, less thanabout 5.0×10⁻⁶M, less than about 1.0×10⁻⁶M, less than about 5.0×10⁻⁷M,less than about 1.0×10⁻⁷M, less than about 5.0×10⁻⁸M, less than about1.0×10⁻⁸ M, less than about 5.0×10⁻⁹M, or less than about 1.0×10⁻⁹M. Ina further aspect, the concentration can be less than about 5.0×10⁻¹° M,for example, less than about 1.0×10⁻¹⁰ M, less than about 5.0×10⁻¹¹M,less than about 1.0×10⁻¹¹M, less than about 5.0×10⁻¹² M, less than about1.0×10⁻¹²M, less than about 5.0×10⁻¹³M, less than about 1.0×10⁻¹³ M,less than about 5.0×10⁻¹⁴M, less than about 1.0×10⁻¹⁴M, less than about5.0×10⁻¹⁵ M, or less than about 1.0×10⁻¹⁵M.

The disclosed methods can provide real-time, free-solution detection ofmolecular interactions with very low sample volume requirements. Thatis, in one aspect, the invention relates to a free-solution analyticalmethod for detecting molecular interactions comprising the step ofdetecting a molecular interaction between two non-immobilized analytes,wherein at least one of the analytes is present during the determinationin a solution with a volume in the detection zone of less than about 500μL. In various further embodiments, the sample volume can be less thanabout 250 μL, for example, less than about 100 μL, less than about 10μL, less than about 1 μL, less than about 500 nL, less than about 250nL, less than about 100 nL, less than about 10 nL, less than about 1 nL,less than about 500 pL, less than about 250 pL, or less than about 100pL.

In a further aspect, the first and/or second sample comprises anadditive. In a still further aspect, the additive is selected from analcohol, an acid, a base, a high refractive index solvent, a surfactant,and an intercalating agent. In yet a further aspect, the alcohol isdeuterated. In an en even further aspect, the alcohol is fluorous.

In a further aspect, the disclosed methods further comprise the step ofdetermining a change in refractive index. In a still further aspect, thechange in refractive index is at least about 10⁻³ RIU. In yet a furtheraspect, the change in refractive index is at least about 10⁻⁴ RIU. In aneven further aspect, the change in refractive index is at least about10⁻⁵ RIU. In a still further aspect, the change in refractive index isat least about 10⁻⁶ RIU. In yet a further aspect, the change inrefractive index is at least about 10⁻⁷ RIU. In an even further aspect,the change in refractive index is at least about 10⁻⁸ RIU.

In a further aspect, the disclosed methods further comprise the step ofperforming a chromatographic separation and/or an electrophoreticseparation on the sample before, during, or after the determining thedetermining step. In a still further aspect, the method furthercomprises the step of performing a chromatographic separation or anelectrophoretic separation on the sample prior to the determining thedetermining step. In yet a further aspect, the method further comprisesperforming a chromatographic separation or an electrophoretic separationon the sample during the determining the determining step. In an evenfurther aspect, the method further comprises performing achromatographic separation or an electrophoretic separation on thesample after the determining the determining step. In a still furtheraspect, the method further comprises performing a chromatographicseparation and an electrophoretic separation on the sample prior to thedetermining the determining step. In yet a further aspect, the methodfurther comprises performing a chromatographic separation and anelectrophoretic separation on the sample during the determining thedetermining step. In an even further aspect, the method furthercomprises performing a chromatographic separation and an electrophoreticseparation on the sample after the determining the determining step.

E. Methods for Determining a Characteristic Property of a Sample

In one aspect, disclosed are methods for determining a characteristicproperty of a sample, the method comprising the steps of: (a) providinga refractive index sensor for reception of a fluid sample to beanalyzed; (b) introducing a fluid sample to be analyzed onto the sensor;(c) interrogating the fluid sample with light; (d) detecting the lightafter interaction with the fluid sample, wherein the detected light isnot backscattered; and (e) determining the characteristic property ofthe sample. In a further aspect, the fluid sample to be analyzedcomprises an analyte. In a still further aspect, the analyte isnon-immobilized. In yet a further aspect, the analyte is unlabeled.

In one aspect, disclosed are free-solution analytical method comprisingdetecting a molecular change, wherein the detection is performed byrefractive index sensing other than backscattering interferometry. In afurther aspect, the detection is performed by refractive index sensingother than forward scattering or side scattering interferometry. In astill further aspect, detecting a molecular change comprises determiningthe degree of polymerization, protein folding, protein aggregation,blood oxygenation, the conformational state of an ion channel ormembrane protein, or the hydration state of an ion Chanel or membraneprotein.

In a further aspect, the light is not scattered.

In a further aspect, the refractive index sensor comprises a substratehaving a first channel formed therein for reception of a fluid sample tobe analyzed and wherein the sample is introduced into the channel.Generally, the substrate and channel can comprise any material suitablefor containing and providing a sample for analysis and capable of beinginterrogated by light. In one aspect, the substrate and channel togethercomprise a capillary tube. In a further aspect, wherein the substrateand channel together comprise a microfluidic device.

In a further aspect, a channel is formed in the substrate and the methodfurther comprises the steps of: (a) introducing a reference sample inthe second channel; (b) determining a characteristic of the referencesample; and (c) employing the characteristic of the reference sample tocompensate for background interference effects in the determination ofthe characteristic of the sample in the first channel.

In a further aspect, the substrate has a channel formed therein with agenerally hemispherical cross sectional shape. In a still furtheraspect, the channel is formed with first and second curved potions, eachcurved portion defining a 90° arc, and a first flat portion connectingthe first and second curved portions.

In a further aspect, the sample is positioned inside a channel formed ina substrate, the channel has a longitudinal direction and a transversedirection, and the light is elongated in the longitudinal direction ofthe channel. In a still further aspect, the light is incident on atleast a portion of the channel greater than 4 mm in length along thelongitudinal direction.

In various aspects, the light source generates an easy to align opticalbeam that is incident on the etched channel for generating scatteredlight. In a further aspect, the light source generates an optical beamthat is collimated, such as, for example, the light emitted from a HeNelaser. In a still further aspect, the light source generates an opticalbeam that is not well collimated and disperses in, for example, aGaussian profile, such as that generated by a diode laser.

Typically, two types of lasers can be employed. In various aspects, onelaser (the diode) creates a laser beam that is elongated in thelongitudinal direction of the channel. In further aspects, the other(HeNe) creates a laser beam that is not elongated longitudinally alongthe length of the channel, but can be later elongated longitudinallyalong the length of the channel by beam-stretching optics. These methodscan both achieve the same end of an elongated beam impinging upon thechannel, but do so through different means. It can be noted that, incertain aspects, when the diameter of the laser beam is the same as thethickness of the glass chip, new interference phenomena can arise. Thiscan be avoided by selecting the width of the beam to be smaller than thethickness of the glass chip (0.8 mm width laser and 1.7 mm thicknessglass chip).

In a further aspect, a single light beam is incident upon the substrate.

In a further aspect, the light beam has a substantially uniformintensity profile across at least a portion of the plurality of discretezones. In a yet further aspect, the light beam has a substantiallyGaussian intensity profile in the axis perpendicular to the zones. In astill further aspect, the portion of the light beam impinging thechannel has an elongated intensity profile.

In various aspects, the light beam is incident on at least a portion ofthe channel greater than 4 mm in length along the longitudinaldirection. In a further aspect, the light beam is incident on at least aportion of the channel greater than 5 mm of length of the channel in thelongitudinal direction. In a still further aspect, the light beam isincident on at least a portion of the channel greater than 6 mm oflength of the channel in the longitudinal direction. In yet a furtheraspect, the light beam is incident on at least a portion of the channelgreater than 7 mm of length of the channel in the longitudinaldirection. In an even further aspect, the light beam is incident on atleast a portion of the channel greater than 8 mm of length of thechannel in the longitudinal direction. In a still further aspect, thelight beam is incident on at least a portion of the channel greater than9 mm of length of the channel in the longitudinal direction. In yet afurther aspect, the light beam is incident on at least a portion of thechannel greater than 10 mm, 12 mm, 14 mm, 16 mm, 18 mm, or 20 mm oflength of the channel in the longitudinal direction.

In a further aspect, at least a portion of the light beam incident onthe channel covers at least two discrete zones. In a still furtheraspect, at least a portion of the light beam is incident on the channelsuch that the intensity of the light on each of at least two zones isthe same or substantially the same. In yet a further aspect, at least aportion of the light beam is incident on the channel such that the eachof the zones along the channel receive the same or substantially thesame intensity of light. For example, a light beam having a Gaussianintensity profile can be incident on a channel such that at least twozones along the channel are within the peak of the intensity profile,receiving the same or substantially the same intensity of light. In aneven further aspect, the portion of the light beam incident on thechannel can have a non-Gaussian profile, such as, for example, a plateau(e.g., top-hat). The portion of the light beam in the wings of theGaussian intensity profile can be incident upon other portions of thechannel or can be directed elsewhere.

In a further aspect, variations in light intensity across zones ofinterest can result in measurement errors. In a still further aspect, ifportions of a light beam having varying intensity are incident uponmultiple zones of a channel, a calibration can be performed wherein theexpected intensity of light, resulting interaction, and scattering isdetermined for correlation of future measurements.

The light source can comprise any suitable equipment and/or means forgenerating light, provided that the frequency and intensity of thegenerated light are sufficient to interact with a sample and/or a markercompound and provide elongated fringe patterns as described herein.Light sources, such as HeNe lasers and diode lasers, are commerciallyavailable and one of skill in the art could readily select anappropriate light source for use with the systems and methods of thepresent invention.

In a further aspect, the light beam is directed from a laser formedintegrally on the substrate. In a still further aspect, thephotodetector is formed integrally on the substrate.

In a further aspect, the characteristic property comprises the index ofrefraction of the sample. In a still further aspect, the characteristicproperty comprises the temperature of the sample.

In a further aspect, the characteristic property to be determined iswhether first and second biochemical functional species bind with oneanother, and the step of introducing a sample to be analyzed onto thesensor comprises introducing the first biochemical functional speciesinto the channel and introducing the second biochemical functionalspecies into the channel to facilitate a binding reaction between thefirst and second biochemical species.

In a further aspect, the first and second biochemical functional speciesare introduced sequentially. In a still further aspect, the first andsecond biochemical functional species are introduced simultaneously.

In a further aspect, the first biochemical functional species is allowedto interact with the second biochemical functional species prior tointroducing the first and/or second biochemical species onto the sensor.In a still further aspect, the first biochemical functional species isallowed to interact with the second biochemical functional species afterintroducing the first and/or second biochemical species onto the sensor.

In a further aspect, the first and second biochemical functional speciesare selected from the group comprising complimentary strands of DNA,complimentary proteins, and antibody antigen pairs.

In a further aspect, the substrate is selected to be formed from PDMS.

In a further aspect, the laser beam is selected to have a diameter of 2mm or less. In a still further aspect, the channel, when present, isselected to have a width that is no larger than the diameter of thelaser beam.

In a further aspect, the refractive index sensor comprises a prism.

What is claimed is:
 1. A free-solution analytical method comprising detecting molecular interactions between a first non-immobilized analyte and a second non-immobilized analyte, wherein the detection is performed by refractive index sensing other than backscattering interferometry, or by circular dichroism.
 2. The method of claim 1, wherein the detection is performed by refractive index sensing other than forward scattering or side scattering interferometry.
 3. The method of claim 1, wherein both analytes are unlabeled.
 4. The method of claim 1, wherein at least one of the analytes is present in an amount of less than about 1.0×10⁻³ M.
 5. The method of claim 1, wherein detecting comprises one or more of: (a) determining an equilibrium constant, a dissociation constant, a dissociation rate, or an association rate; (b) calculating a change in hydrodynamic volume, entropy, or enthalpy; (c) determining the concentration of the first and/or second analyte; (d) identifying the presence of the first and/or second analyte; and (e) identifying the presence of a third analyte.
 6. The method of claim 1, wherein refractive index sensing is via a hand-held refractive index sensing device.
 7. The method of claim 1, wherein refractive index sensing is via a RI detectors based on the angle of deviation method of measurement, a RI detectors based on the Fresnel method of RI measurement, a Christiansen effect detector, an interferometer detector, or a differential refractometer detector.
 8. A free-solution analytical method comprising the steps of: (a) providing a refractive index sensor for reception of a fluid sample to be analyzed; (b) introducing a first sample comprising a first non-immobilized analyte to be analyzed and a second sample comprising a second non-immobilized analyte to be analyzed onto the sensor, wherein the first analyte is allowed to interact with the second analyte; (c) interrogating the fluid sample with light; (d) detecting the light after interaction with the fluid sample, wherein the detected light is not backscattered; and (e) detecting a molecular interaction between the first and second analyte.
 9. The method of claim 8, wherein the sample is positioned inside a channel formed in a substrate, the channel has a longitudinal direction and a transverse direction, and the light is elongated in the longitudinal direction of the channel.
 10. The method of claim 9, wherein the light is incident on at least a portion of the channel greater than 4 mm in length along the longitudinal direction.
 11. The method of claim 8, wherein the light is not scattered.
 12. The method of claim 8, wherein the first and/or second analyte is a complex.
 13. The method of claim 8, wherein the molecular interaction is the formation of one or more covalent bonds, electrostatic bonds, hydrogen bonds, or hydrophobic interactions.
 14. The method of claim 8, wherein the first and/or second analyte is one or more of an antibody, an antigen, a protein, a small molecule, a drug, a receptor, a cell, an oligonucleotide, a carbohydrate, an enzyme, a substrate, a DNA, an aptamer, a RNA, a nucleic acid, a biomolecule, a molecular imprint, a protein mimetic, an antibody derivative, a lectin, a cell membrane, an ion, a virus particle, a bacteria, and a micro-RNA.
 15. The method of claim 8, wherein the molecular interaction is a binding event between one or more of antibody-antigen, protein-protein, small molecule-small molecule, small molecule-protein, drug-receptor, antibody-cell, virus-cell, virus-protein, bacteria-cell, bacteria-protein, virus-DNA, virus-RNA, bacteria-DNA, bacteria-RNA, protein-cell, oligonucleotide-cell, carbohydrate-cell, cell-cell, enzyme-substrate, protein-DNA, protein-aptamer, DNA-DNA, RNA-DNA, DNA-RNA, protein-RNA, small molecule-nucleic acid, biomolecule-molecular imprint, biomolecule-protein mimetic, biomolecule-antibody derivatives, lectin-carbohydrate, biomolecule-carbohydrate, small molecule-cell membrane, ion-protein, and protein-protein.
 16. The method of claim 8, wherein the molecular interaction lacks a change in mass.
 17. The method of claim 8, wherein the first and second sample are introduced simultaneously.
 18. The method of claim 8, wherein the first and second sample are introduced sequentially.
 19. A free-solution analytical method comprising determining the degree of polymerization, protein folding, protein aggregation, blood oxygenation, the conformational state of an ion channel or membrane protein, or the hydration state of an ion Chanel or membrane protein, and wherein the determination is performed by refractive index sensing.
 20. The method of claim 19, wherein at least one of the analytes is unlabeled.
 21. A system comprising a refractive index sensor for detecting molecular interactions between a first non-immobilized analyte and a second non-immobilized analyte, and a pressure change compensator.
 22. The system of claim 21, wherein the sensor comprises a channel formed in a substrate, the channel has a longitudinal direction and a transverse direction, and a light source for generating a light, wherein the light is elongated in the longitudinal direction of the channel.
 23. The system of claim 21, wherein both analytes are unlabeled.
 24. The system of claim 21, wherein at least one of the analytes is present in an amount of less than about 1.0×10⁻³ M.
 25. A free-solution analytical method comprising detecting a molecular change, wherein the detection is performed by refractive index sensing other than backscattering interferometry.
 26. The method of claim 25, wherein detecting a molecular change comprises determining the degree of polymerization, protein folding, protein aggregation, blood oxygenation, the conformational state of an ion channel or membrane protein, or the hydration state of an ion chanel or membrane protein.
 27. The method of claim 25, wherein the detection is performed by refractive index sensing other than forward scattering or side scattering interferometry. 